The organotypic, or "raft," tissue culture system involves culturing epithelial cells at an air-liquid interface so that the cells fully differentiate in a manner similar to that found in vitro. Although initially developed for epidermal cells, the method has been used in this laboratory to provide an in vitro mimic of cell differentiation using foreskin, uterine cervical, vaginal and oral epithelial cells. While normal, e.g. non-infected, human cells display characteristic terminal differentiation on rafts, the phenotype of cells expressing viral genes can range from normal through dysplastic to malignant. This has been particularly well-demonstrated in histological preparations from rafts prepared with cells expressing human papillomavirus (HPV) genes. The raft preparations are also sensitive indicators to toxicity resulting from abnormal gene expression and/or constituents of the medium. The organotypic culture method will provide an ideal approach to the evaluation of the effects of candidate microbicides on cell phenotype, host cell gene expression and viral gene expression. We propose to use vaginal and other human genital area epithelial cells to assess the effects of microbicide compounds on cell viability, cell growth and differentiation. We will investigate the response of viral gene expression, using HPV and HSV-2 gene constructs, and possible effects on virus infectivity. Although HPV virions have not been grown in quantity in vitro, the preparation of recombinant viral capsids will allow studies of receptors, etc.,for that virus to proceed in the near future. We will also examine the effects of the microbicides on the phenotype of cells immortalized by HPV-16 or -18 as a further measure of the inhibition of cellular response to infection. This project will contribute to the overall program by providing a cell biology and molecular biology approach to the evaluation of microbicides, using differentiating relevant human cells in an in vitro setting. The project will interact with Project 1 by establishing primate organotypic cell cultures for further evaluation and comparison with human cells and with project 4, using lipid mixtures in antiviral experiments. This project is dependent upon cooperation with the Chlamydia Core (B) and the Clinical Core (D) for cell, organisms, and tissues.